Efficacy of Modified Papanicolaou stain for demonstration of keratin in paraffin embedded tissue sections

INTRODUCTION

Epithelial tissues form a barrier between the body and the environment. Depending on location, they perform various functions, but always serve to protect the internal tissues from environmental stresses, chemical damage, and bacterial infection¹ .

A critical aspect of stratified keratinizing epithelia is that the cells undergo a terminal differentiation program that results in the formation of a mechanically resistant and toughened surface composed of cornified cells that are filled with keratin filaments and lack nuclei and cytoplasmic organelles. In these squames, the cell membrane is replaced by a proteinaceous cornified envelop that is covalently cross-linked to the keratin filaments, providing a highly insoluble yet flexible structure that protects the underlying epithelial cells¹ .

Keratins are frequently the most abundant cellular proteins. They constitute the major component of the cytoskeleton of all epithelia; these intermediate filaments provide mechanical support for the cells and nucleus. Keratins have a number of distinct advantages for use as marker proteins to differentiate epithelial tumors from mesenchymal t u m o r s b o t h h i s t o l o g i c a l l y a n d immunohistochemically. In establishing a definitive diagnosis, it is sometimes advantageous to demonstrate histologically the degree of keratinization or the presence and / or absence of keratin through the application of a stain which discerns keratin² .

In routine Haematoxylin and Eosin (H-E) staining, structures like collagen, amyloid, muscle, keratin and other extra cellular and intracellular secretions stain eosinophilic where differentiating one from other is difficult. For example the histological demonstration of keratin is important in assessing the degree/ pattern of differentiation for squamous cell carcinoma.

In the past, Ayoub-Shklar (A/S), Dane - Herman method, Schiff reagent after oxidation with performic acid, aldehyde-fuchsin, levafix red violet have been used to stain keratin. All these have advantages and disadvantages.

Papanicolaou (PAP) stain is a routinely used staining technique, commonly available in Oral Pathology laboratories. It is a multichromatic staining technique which is used to differentiate cells in smear preparations for various body secretions³ . The main use of orange G6 in the papanicolaou stain is to stain keratin⁴. Superficial cells with high content of keratin stain yellow-orange hue and parabasal cells stain green to blue in color³ . 

Johnson and Klein, in 1956 reported the application of the papanicolaou stain to paraffin embedded sections, for demonstration of keratin⁵ . Richard P.Elzay has reported the Modification of Papanicolaou stain by adding Phloxine-B on paraffin embedded sections to demonstrate keratin² . Phloxine-B, a red acid dye is a derivative of fluroscein with distinctly bluish shade⁶. It is used to stain mucin, prekeratin and keratin which appear distinctly red in color. 

Although the presence of keratin protein can be detected very specifically by imunohistochemical methods on paraffin embedded tissue sections, the antikeratin-antisera are not economical and are time consuming^2. Hence this study was designed to demonstrate the efficacy of Modified Papanicolaou (Mod PAP) stain with phloxine - B in tissues where keratin was present. The efficacy was compared with routine Haematoxylin and Eosin, the commonly used staining methods so as to device an optimal staining technique for keratin that would be specific, easy, cost and time effective. 

MATERIALS AND METHODS

Since the study was planned to stain keratin in tissue sections, Haematoxylin and Eosin stained sections were reviewed for the presence of keratin in known keratin containing tissue sections and slides were selected.

The study group included: 

• 17 cases of Verrucous Carcinoma (VC) 
• 15 cases of Normal Keratinized Oral Mucosa (NKOM)
• 8 cases of Keratinized Odontogenic Keratocyst (OKC).

A Total Number of 40 paraffin embedded tissue blocks were retrieved from the archives for our study.

Method of collection of Data:

Two sections of 4 micron thickness paraffin embedded sections were taken. Sections were stained with 1) Haematoxylin and Eosin 2) Modified Papanicolaou stain respectively.

Procedure for Modified Papanicolaou stain:

• Solutions used: 
• Harris's hematoxylin
• Orange G6
• Eosin – azure 
• Phloxine – B (1% aqueous C.I no 45410) 

Procedure for staining:

1. Deparaffinize sections through 2 changes of xylene, absolute alcohol, and 95% alcohol to water wash.

2. Stain in Harris's hematoxylin for 6min

3. Rinse in two changes of tap water and dip in acid alcohol

4. Rinse thoroughly in tap water and dip in lithium carbonate

5. Wash in running tap water for 10min, then rinse in distilled water

6. Stain in phloxine –B for 5min

7. Rinse in distilled water and dehydrate

8. Stain in Orange G6 for 5min

9. Rinse in 95% alcohol

10.Stain in eosin azure for 4min

11.Rinse in 95% alcohol complete dehydration in absolute alcohol

12.Clear in xylene and mount.

Stained sections were evaluated by three oral pathologists independently and consensus was taken when required. The results were analysed for efficacy of staining technique and examined for:

1. Type o f Surf a c e k e r atin (pa r a o r orthokeratinized)

2. Pattern of keratin staining (whether uniform or patchy).

Results were tabulated and statistically analyzed using Chi square test. P value ≤ 0.05 was considered to be statistically significant. 

RESULTS

In our study H & E and Modified PAP stain showed clear, distinct keratin as pink and magenta pink respectively.

Both the staining techniques showed similar results for “Type of Surface keratin” in Normal Mucosa, Verrucous Carcinoma, Odontogenic Keratocyst (Table – 1, Fig 1-6).

Of 15 cases of NKOM, in H-E stain, 14 cases showed only parakeratinized layer and one showed both para and orthokeratinized layer. In Mod PAP stain, 13 cases showed only parakeratinized layer and only 2 cases showed both para and orthokeratinized layer. Results were not statistically significant when H- E was compared with Modified PAP (Table 1, Figure 1 & 2).

Of 8 cases of OKC, in H-E stain, 6 cases showed only parakeratinized layer and 2 showed both para and orthokeratinized layer. In Mod PAP stain, 5 cases showed only parakeratinized layer 1 case showed only ortho and 2 showed both para and orthokeratinized layer. Results were not statistically significant when H- E was compared with Modified PAP (Table 1, Figure 3 & 4).

Of 17 cases of VC both H-E and Modified PAP showed similar results i.e. all 17 cases showed only parakeratinized epithelium. Results were not statistically significant when H& E was compared with Modified PAP (Table 1, Figure 5 & 6).

When evaluated for “Pattern of staining”, in Normal Mucosa, Verrucous Carcinoma, Odontogenic Keratocyst both staining technique showed similar results (Table – 2, Figure 1-6).

Of 15 cases of NKOM, in both H-E stain and Modified PAP, all 15 cases showed uniform staining pattern. Results were not statistically significant (Table - 2, Figure 1 & 2).

Of the 8 cases of OKC, in H-E stain, all 8 cases showed uniform staining pattern and in Mod PAP Stain, 7 cases showed uniform staining pattern and one case showed patchy staining pattern. Results were not statistically significant (Table 2, Figure 3 & 4).

Of the 17 cases of VC, in H-E stain all 17 cases showed uniform staining pattern. And in Modified PAP 15 cases showed uniform and two cases showed patchy stain. Results were not statistically significant (Table 2, Figure 5 & 6).

DISCUSSION

The Oral Epithelium represents the primary barrier between oral environment and the deeper tissues. It is lined by stratified squamous epithelium and consists of cells tightly attached to one another and arranged in a number of distinct layers or strata. It maintains its structural integrity by a system of continuous cell renewal⁷. 

The epithelial surface of the masticatory mucosa, such as that of the hard palate and gingiva is flexible, tough and resistant to abrasion. This is caused by the formation of a surface layer of keratin and the process of maturation is called keratinization. Keratin constitutes the major component of cytoskeleton of all epithelia and provides a mechanical support for the cells and nucleus⁷. 

Keratin plays an important role as a marker protein in establishing a definitive histological diagnosis, like for e.g.; in grading of Squamous Cell Carcinoma² , to differentiate between the epithelial and mesenchymal tumors and in certain conditions like when the epithelial component may be sparse and may be identified only by the presence of keratin reactivity.

Special stains are the stains that are used to visualize specific tissues and cellular structures. These are the dyes that bind to the cellular components either physically or by chemical bonds. Ayoub-shklar and Dane Herman method are special histochemical stains used to stain keratin specifically are all special histochemical stains used to stain keratin specifically. These stains may highlight small foci of overt epithelial differentiation that sometimes is missed in routine H-E staining.

Papanicolaou stain is a routinely used cytopathological stain available in most of the Oral Pathology laboratories. Johnson and Klein, in 1956 reported the application of the Papanicolaou stain to paraffin embedded sections, for demonstration of keratin⁵. Later Richard P.Elzay modified the routine Papanicolaou stain by adding phloxine-B, which is a red acid dye that stains prekeratin and keratin distinctly red in color on paraffin embedded sections. Thus the Modified PAP stain can be used to stain keratin specifically².

Thus, the aim of the present study was to stain the known keratin containing paraffin embedded tissue with Modified Papanicolaou stain, and H-E stain and to compare the efficacy of Modified Papanicolaou stain with that of H-E staining technique, so as to design a staining procedure which is easy and effective for keratin.

In our study both H-E and modified PAP staining techniques showed similar findings in staining the “Type of Surface keratin” in NKOM, OKC, and VC. In Majority of cases both parakeratinized and orthokeratinized layers were distinctly stained where H-E staining procedures imparted keratin as pink, and Modified PAP stain as Magenta pink in colour (Table -1 Fig 1-6). 

When evaluated for “Pattern of staining”, in NKOM, OKC, and VC in both stainig procedures majority of cases showed uniform staining pattern. (Table – 2, Fig 1- 6).

Our study was in consistent with study of Johnson and Klein⁵ who used papanicolou stain on paraffin embedded tissues sections. In a similar study done by Richard.P.Elzay et al.², the parakeratinized layer did not take up uniform stain in modified PAP stain when compared to orthokeratinized layer. 

These results were not consistent with our study where the parakeratinized layer was distinctly stained as magenta pink in color.

Although the results of our study were not statistically significant modified PAP has more advantage over routine H-E which is gold standard and not specific for keratin. Whereas, modified PAP is a polychrome stain which apart from staining keratin magenta pink, also imparts different hues of colour to structures like Nuclei – Blue, Collagen – Green, Bone – Blue, Muscle – and Erythocytes as Red in colour. Thus the Modified PAP stain can be used to stain keratin specifically on paraffin embedded tissue sections.

CONCLUSION

The present study, to the best of our knowledge, is the first study to document and compare H-E and modified PAP for keratin in Normal keratinized oral mucosa, Odontogenic keratocyst and Verrucous carrcinoma. Hence, our study adds to the limited literature on application of modified PAP stain on paraffin embedded tissue sections to stain Surface keratin.

ACKNOWLEDGMENTS

A sincere thanks to Dr. Srinivas Vanaki, Principal, Head of the Department of Oral Pathology, PMNM Dental College, Bagalkot for his constant support.